The CD70-CAR T cells were generated as our previous publication (22 (link)). Briefly, CD70 and CD19 CAR contain a humanized 41D12-based scFv or FMC63 mAb, respectively, followed by the CD8α-based extracellular spacer and transmembrane domains with 41BB-CD3ζ intracellular signaling domain. CD3+ T cells were purified from peripheral blood mononuclear cells (PBMNCs) of healthy donors and were activated with CD3/CD28 dynabeads (Gibco, USA), which were then transfected with lentivirus encoding CD70 or CD19 CAR with 10 μg/mL polybrene (Sigma, Germany) at a multiplicity of infection of 20 (Sigma, Germany). The CD3/CD28 dynabeads were removed on days 3-5, and the transfected T cells were expanded in AIM-V (Gibco, USA) medium supplemented with 10% FBS (Gibco, Australia), 300 IU/mL IL-2, 5 ng/mL IL-7 and IL-15 for approximately 10 days.
Cd3 cd28 dynabead
Manufactured by Thermo Fisher Scientific
Sourced in United States, Germany, Switzerland, Norway, United Kingdom
CD3/CD28 Dynabeads are a type of magnetic beads coated with antibodies against the CD3 and CD28 T cell surface markers. These beads are used to activate and expand T cells in vitro for various research and therapeutic applications.
Automatically generated - may contain errors
Lab products found in correlation
Interleukin 2 (il 2), by Thermo Fisher Scientific (27 mentions)
Retronectin, by Takara Bio (15 mentions)
Pan t cell isolation kit, by Miltenyi Biotec (12 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (10 mentions)
Celltrace violet, by Thermo Fisher Scientific (9 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (8 mentions)
Rpmi 1640, by Thermo Fisher Scientific (8 mentions)
Carboxyfluorescein succinimidyl ester (cfse), by Thermo Fisher Scientific (8 mentions)
Sodium pyruvate, by Thermo Fisher Scientific (7 mentions)
Glutamax, by Thermo Fisher Scientific (7 mentions)
Aim 5 albumax medium, by Thermo Fisher Scientific (7 mentions)
β mercaptoethanol, by Merck Group (6 mentions)
Pan t cell isolation kit 2, by Miltenyi Biotec (6 mentions)
Hepes, by Merck Group (6 mentions)
Il 15, by Thermo Fisher Scientific (5 mentions)
Ficoll paque plus, by GE Healthcare (5 mentions)
L glutamine, by Thermo Fisher Scientific (5 mentions)
Easysep human t cell enrichment kit, by STEMCELL (5 mentions)
X vivo 15, by Lonza (5 mentions)
Hepes, by Thermo Fisher Scientific (5 mentions)
277 protocols using cd3 cd28 dynabead
1
Generation of CD70-CAR T Cells
2
T Cell Activation and PD-L1 Modulation
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CD8+ T cells were labelled with Cell Trace Violet (CTV) (Thermo Fisher Scientific) for 20 min and rested overnight before being stimulated for 72 hr with CD3/CD28 Dynabeads (Thermo Fisher Scientific). Jurkat.PDCD1 cells were stimulated for 24 hr with CD3/CD28 Dynabeads (Thermo Fisher Scientific). Primary or cell line T cells were co-cultured with HEK293T cells transfected with pcDNA3.1-CD274v1 or pcDNA3.1-CD274-L2A as indicated. Conditioned media from HEK293T.CD274-L2A cells, parental HEK293T cells, B-3T3.CD274-L2A cells, parental B-3T3 cells, IFN-γ-stimulated HBL-1 cells, parental HBL-1 cells, or 10 μg/mL ultra-LEAF anti-human PD-L1 antibody (29E.2A3, Biolegend) were added as indicated.
3
Lentiviral Transduction of Mouse and Human T Cells
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Mouse T cells were activated by adding CD3/CD28 Dynabeads (Thermo Fisher Scientific) according to the manufacturer’s instructions and then transduced with lentiviral supernatants. Lentivirus was then added to retronectin (Takara)-coated non-tissue culture–treated plates to reach a total volume of 400 µL and mixed gently. T cells and virus were centrifuged at 1000 g for 2 hours and then incubated at 37°C. After 3 days of culture, the medium was changed to complete RPMI-1640 medium (10% FBS, 2 mM GlutaMAX, 100 µM β-mercaptoethanol, 1% penicillin/streptomycin, 50 U/mL rmIL-2).
Human T cells were stimulated by CD3/CD28 Dynabeads (Thermo Fisher Scientific) according to the manufacturer’s instructions. On day 2, T cells were plated in non-tissue culture-coated 24-well plates, and polybrene (8 µg/mL) was added to the medium. Lentiviral supernatant was first centrifuged at 1500 g for 2 hours on retronectin (Takara)-coated non-tissue culture-treated plates. T cells were then plated and centrifuged at 1000 g for 20 min and incubated at 37°C. After 3 days, the medium was changed to 45% RPMI-1640% and 45% Click’s medium containing 10% FBS and supplemented with rhIL-2 (50 U/mL), rhIL-7 (10 ng/mL), and rhIL-15 (5 ng/mL).
Human T cells were stimulated by CD3/CD28 Dynabeads (Thermo Fisher Scientific) according to the manufacturer’s instructions. On day 2, T cells were plated in non-tissue culture-coated 24-well plates, and polybrene (8 µg/mL) was added to the medium. Lentiviral supernatant was first centrifuged at 1500 g for 2 hours on retronectin (Takara)-coated non-tissue culture-treated plates. T cells were then plated and centrifuged at 1000 g for 20 min and incubated at 37°C. After 3 days, the medium was changed to 45% RPMI-1640% and 45% Click’s medium containing 10% FBS and supplemented with rhIL-2 (50 U/mL), rhIL-7 (10 ng/mL), and rhIL-15 (5 ng/mL).
4
Murine and Human CD8+ T Cell Culture
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Murine CD8+ T cells were isolated (STEMCELL, 19853) and activated with 1 μg/mL αCD3 (eBioscience, 16-0031-86) and 2.5 μg/mL αCD28 (eBioscience,16-0281-82) for 2 days prior to subculture in the presence of 20 U/mL recombinant murine IL-2 (Peprotech, 50-813-288).
For human T cell culture, frozen negatively isolated CD8+ T cells (Hemacare, PB08NC1) were activated with CD3-CD28 Dynabeads (Gibco, 11131D) for 72 hours in the presence of 20 U/mL IL-2 (Roche, 11011456001) and 5 ng/mL IL-7 (Peprotech, 200-07) and IL-15 (Peprotech, 200-15). Cells were subcultured with fresh media and cytokines.
For human T cell culture, frozen negatively isolated CD8+ T cells (Hemacare, PB08NC1) were activated with CD3-CD28 Dynabeads (Gibco, 11131D) for 72 hours in the presence of 20 U/mL IL-2 (Roche, 11011456001) and 5 ng/mL IL-7 (Peprotech, 200-07) and IL-15 (Peprotech, 200-15). Cells were subcultured with fresh media and cytokines.
5
Isolation and Activation of Human T Cells
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To verify if FITC-YT-16 exerts a functional effect on human T cells, we assessed peptide performance on freshly isolated T cells. Briefly, human peripheral blood mononuclear cells (PBMCs) were isolated from healthy donor’s blood by density gradient centrifugation using Ficoll-Paque PLUS (GE Healthcare, Little Chalfont, Buckinghamshire, UK)). In brief, blood was diluted at 1:1 with phosphate-buffered saline (PBS), layered on Ficoll-Paque and centrifuged at 2000× g for 20 min. PBMCs were re-suspended in phosphate-buffered saline. Isolated T cells were immediately enriched by magnetic microbeads positive selection (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany). For stimulating and activating T cells, isolated T cells were cultured in 6 well-plate in RPMI-1640 medium with 10% fetal bovine serum (FBS), 100 IU/mL penicillin, and 100 µg/mL streptomycin (Gibco, Life Technologies, Grand Island, NY, USA). Then inoculated with human T-activator CD3/CD28 Dynabeads (Gibco, Life Technologies, Grand Island, NY, USA, USA) at a ratio of one bead to one T cell for 24 h, supplemented with 50 IU/mL of recombinant human IL-2 (Novus Biologicals, Littleton, CO, USA).
6
Generation of IL-10Rα-Deficient Murine P14 CD8+ T Cells
7
Transducing T cells with Retroviral Constructs
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T cells isolated from PBMCs were stimulated with CD3/CD28 Dynabeads (Gibco, Cat#11 132D) for 24 hours. T cells were transduced with retroviral constructs containing tdTomato and click beetle red luciferase in RetroNectin (Takara Bio, #Cat T100A/B)-coated six-well plates in the presence of IL-2 (100 IU/mL) and protamine sulfate (4 µg/mL). Transduced T cells were cultured for 8 days before use in animal experiments.
8
Cytolysis Assay for Bispecific Antibodies
9
Murine T Cell Modulation by hUCB-MDSCs
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Splenocytes from NC/Nga mice were seeded at a density of 1 × 106 cells/well into 24-well plates (Falcon, Corning, NY, USA) and stimulated with CD3/CD28 DYNABEADS (Gibco, Thermo Fisher Scientific, Waltham, MA, USA) to analyze T cell proliferation. hUCB-MDSCs were cocultured with spleen cells at ratios of 0.5:1, 0.25:1, and 0.1:1. Thereafter, cell proliferation was assessed using the Cell Trace CFSE Cell Proliferation Kit (Invitrogen, Thermo Fisher Scientific) per the manufacturer’s instructions.
To analyze T-cell differentiation, purified CD4+ T cells from NC/Nga mice were seeded at a density of 3.12 × 105 cells/well into 24-well plates (Falcon) and differentiated using the Th2 differentiation kit (STEMCELL Technologies, Vancouver, Canada) or Th17 differentiation kit (R&D systems, Minneapolis, MN, USA). hUCB-MDSCs were cocultured with CD4+ T cells at ratios of 1:1, 0.5:1, and 0.25:1. Th2 and Th17 cell cultures were incubated for 6 and 5 days, respectively, according to the manufacturer’s instructions.
To analyze T-cell differentiation, purified CD4+ T cells from NC/Nga mice were seeded at a density of 3.12 × 105 cells/well into 24-well plates (Falcon) and differentiated using the Th2 differentiation kit (STEMCELL Technologies, Vancouver, Canada) or Th17 differentiation kit (R&D systems, Minneapolis, MN, USA). hUCB-MDSCs were cocultured with CD4+ T cells at ratios of 1:1, 0.5:1, and 0.25:1. Th2 and Th17 cell cultures were incubated for 6 and 5 days, respectively, according to the manufacturer’s instructions.
10
Adoptive Transfer of Ribociclib-Treated CD8+ T Cells
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Spleens and lymph nodes were harvested from TRP1high CD45.1+ mice. They were crushed through 40-μm cell strainers in PBS. CD8+ T cells were isolated by negative selection using an EasySep Mouse CD8+ T cell Isolation Kit (StemCell #19853). The cells were cultured in RPMI complete containing 100 U/ml human IL-2 (Peprotech 200-02-250UG) and CD3/CD28 Dynabeads (#11456; Gibco) with or without ribociclib at 200 nM. After 48 h, 2 × 106 CD45.1+ CD8+ T cells in 150 μl of sterile PBS were transferred by tail vein injection into sex-matched CD45.2+ C57BL/6 recipient mice, with half of the mice receiving cells that had been activated in the presence of ribociclib. For the longitudinal monitoring of cell persistence after transfer, mice were bled in the indicated intervals, and flow cytometry was performed.
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